Our objectives coalesce in efforts to develop an efficient in vitro system for the packaging of DNA sequences into adenovirus capsids. If successful, the resulting particles could be used to attain a nearly synchronous, high multiplicity transfection of susceptible cells by homogeneous and defined, but not necessarily viral DNA sequences. The potential for novel gene expression in such cells may be assessed with regard to the organization of the transfecting DNA and the species, differentiated state or the tissue origin of the cells. The rationale for such an analysis would parallel the study of early viral gene expression in productive or abortive adenovirus infections of animal cells in culture. In pursuit of this long range objective, we have cloned and rearranged DNA from the left end of the adenovirus type 3 (subgroup B) in plasmid pBR322. The cloned sequences represent the region required for polar, left end specific packaging of viral DNA in vivo, the genes for early region Ia/Ib and peptides IVa2 and IX, and the promotor for the major late viral transcription unit. These sequences will be subjected to site-directed deletion and nucleotide alterations in order to define their respective functional domains in assays of DNA packaging and transcription in vitro. DNA packaging will be studied in extracts of arginine deprived, adenovirus infected cells using recently characterized adenovirus empty capsid:DNA complexes. Specific initiation of transcription in vitro will follow the recently described supplementation of purified RNA polymerase II with cytosol (S100) extracts of uninfected cells. These studies should facilitate the design of a cloning vector with the capacity for packaging DNA into adenovirus capsids in vitro and transcription of the inserted DNA sequences under viral promotor control in vivo. We also propose to further develop and exploit the generalized methods for site-directed mutagenesis which resulted from our earlier studies of branched DNA molecules. These methods and the in vitro studies described above should bear considerable impact on future efforts to probe, analyze and possibly modify unique gene expression in eukaryotic cells.